The dynamic remodeling of the actin cytoskeleton is a critical component of nearly all aspects of cell motility, including migration of healthy and metastatic cells and host-pathogen interactions. The rapid remodeling of actin filament networks in cells proceeds through their interactions with actin binding proteins. The overall goal of this proposal is to provide structural and functional understanding of these interactions. Key modulators of actin filaments dynamics in cells are members of the actin-depolymerizing factor (ADF)/cofilin family of proteins. They increase the turnover of actin filaments by severing and depolymerizing them, and also stabilizing them under some conditions. Our past work contributed to the current description of cofilin's action on actin filaments and led to the allosteric, cooperative model of their severing. The work proposed in Aim 1 will bring together multiple approaches, including cryo-electron microscopy, crystallography, fluorescence and electron paramagnetic resonance spectroscopies, fluorescence imaging, mutational work, chemical cross-linking and mass spectrometry methods, to provide structural understanding of the mechanism of cofilin's interactions with actin. The goal will also be to clarify at a structural level the different actin filament severing activities of cofilin isoforms. The severing activity of cofilin is highly regulated in cells, and there is mounting evidence for an important role of coronin in that regulation. The work proposed in Aim 2 will determine the structures of coronin-actin complexes by high resolution cryo-electron microscopy. The goal is to clarify the ATP and ADP dependent differences in the regulation of cofilin function by coronin using multi-method approaches, similar to those listed in Aim 1. The interest in the general understanding of actin filaments severing in cells brings to our focus - in Aim 3 - the role of metavinculin in actin cytoskeleton dynamics. Vinculin and metavinculin connect the actin cytoskeleton to cell membranes in muscle cells, but recent findings show actins filaments severing by the tail domain of metavinculin. The proposed work will determine the high resolution structure of actin- metavinculin complexes by electron microscopy. The parallel goal is to obtain from solution studies a detailed description of the changes in actin filament structure and dynamics caused by metavinculin, and in particular at the interprotomer interaction sites that determine filament stability. The work proposed in Aim 4, on the neuronal actin binding protein debris, is motivated by drebrin's critical role in the formation and function of dendritic spines, which communicate with the synapses of axons. The proposed work will clarify structural and functional aspects of drebrin interactions with actin filaments and drebrin's effect on actin filaments nucleation, branching, severing, dynamics, and structure - in synergy or competition with other neuronal actin binding proteins. The emphasis will be on the changes in the interprotomer contact regions in actin filaments and on mapping the interaction interface on both proteins by cross-linking and mass spectrometry methods. PUBLIC HEALTH RELEVANCE: Our investigation of the mechanism of cofilin and coronin action on actin filaments will deepen the understanding of actin dynamics and remodeling, leading to potential therapeutic intervention in metastatic processes. The goal of clarifying metavinculin-actin interactions and the structure of their complexes is to contribute to better understanding of dilated cardiomyopathy and other disease processes in muscle cells. Drebrin is a critical component of actins cytoskeleton in neuronal cells and our work on its interactions with actin can reveal potential routes for intervention in the diseased state of these cells.